reduction of perioperative inflammatory reaction with exogenous...
TRANSCRIPT
Reduction of perioperative inflammatory reaction with exogenous methane
Ph.D. Thesis
Gábor Bari M.D.
Supervisors: Gabriella Varga Ph.D.
Dániel Érces M.D., Ph.D.
Institute of Surgical Research
University of Szeged, Hungary
2019
2
LIST OF PUBLICATIONS
Full papers related to the subject of the thesis
1. Bari G, Érces D, Varga G, Szűcs S, Varga Z, Bogáts G, Boros M. Methane
inhalation reduces the systemic inflammatory response in a large animal model of
extracorporeal circulation. Eur J Cardiothoracic Surgery 2019.
doi:10.1093/ejcts/ezy453. IF: 3.5
2. Bari G, Érces D, Varga G, Szűcs S, Bogáts G. A pericardialis tamponád
kórélettana, klinikuma és állatkísérletes vizsgálati lehetőségei [Pathophysiology,
clinical and experimental possibilities of pericardial tamponade]. Orv Hetil. 2018.
159:163–167 IF: 0.3
3. Bari G, Szűcs S, Érces D, Ugocsai M, Bozsó N, Balog D, Boros M, Varga G.
A cardiogen sokk modellezése pericardialis tamponáddal [Experimental model for
cardiogenic shock with pericardial tamponade]. Magy Seb. 2017. 70:297–302 IF: 0
4. Szűcs S, Bari G*, Ugocsai M, Lashkarivand RA, Lajkó N, Mohácsi A, Szabó
A, Kaszaki J, Boros M, Érces D, Varga G. Detection of intestinal tissue perfusion by
real-time breath methane analysis in rat and pig models of mesenteric circulatory
distress. Crit Care Med 2019. (*equal contribution, accepted for publication) IF: 6.6
5. Bari G, Szűcs S, Érces D, Boros M, Varga G. Experimental pericardial
tamponade - translation of a clinical problem to its large animal model. Turk J Surg.
2018. 34(3):205–211 IF: 0.1
Abstracts related to the subject of the thesis
1. Bari G, Varga G, Szűcs S, Gules M, Kaszaki J, Boros M, Érces D. Methane
inhalation decreases mucosal mast cell degranulation in a large animal model of
cardiogenic shock. Abstract Book, 17th Congress of the European Shock Society, Paris,
13–15 September 2017.
2. Bari G, Érces D, Szűcs S, Gules M, Gyaraki P, Szilágyi ÁL, Hartmann P, Boros
M, Varga G. Improved platelet function after methane inhalation in a large animal
model of obstructive circulatory shock. Abstract Book, 53rd ESSR Congress, Madrid,
2018, p. 87 (https://www.essr2018.com/abstracts)
3
Full papers not related to the subject of the thesis
1. Csepregi L, Bari G, Bitay M, Hegedűs Z, Varga S, Iglói G, Szabó-B A,
Hartyánszky I, Bogáts G. Kezdeti tapasztalatok a Sorin Perceval S aorta biológiai
billentyű beültetésével [Early experiences with the Sorin Perceval S artificial biological
valve]. Magy Seb. 2016. 69:54–57
2. Bari G, Csepregi L, Bitay M, Bogáts G. A ministernotomia szerepe az
aortabillentyű-sebészetben [The role of mini-sternotomy in aortic valve surgery]. Orv
Hetil. 2016. 157:901–904.
3. Bari G, Bogáts G, Pálinkás E, Badó A, Tiszlavicz L, Kallai A, Vörös E,
Pálinkás A. Bal kamrai kompressziót okozó óriás pericardialis ciszta [Giant pericardial
cyst compressing the left ventricle]. Medicina Thoracalis 2017. 70
4. Bogáts G, Hegedűs Z, Bitay M, Csepregi L, Szabó-Biczók A, Varga S, Iglói G,
Bari G. Coronary artery bypass grafting on the beating heart: 13-year experience in a
single center. J Cardiothoracic Surgery 8: S1 p. O172 (2013)
4
LIST OF ABBREVIATIONS
ACT activated clotting time
ANP atrial natriuretic peptide
CO cardiac output
CLSEM confocal laser scanning endomicroscope
CPB cardiopulmonary bypass
CTMC connective tissue mast cells
ECC extracorporeal circulation
ECMO extracorporeal membrane oxygenation
FcεRI high-affinity IgE receptor
HR heart rate
I-R ischaemia-reperfusion
IBDs inflammatory bowel diseases
ICAM-1 intercellular adhesion molecule 1
IgE immunoglobulin E
Il-4 interleukin-4
Il-5 interleukin-5
MAP mean arterial pressure
MMC mucosal mast cells
MMP matrix metalloproteinase
NAD+ nicotinamide adenine dinucleotide
NOS nitric oxide synthase
PiCCO pulse contour cardiac output
PMN polymorphonuclear
PT pericardial tamponade
RA renal artery
RAA renin-angiotensin-aldosterone
ROS reactive oxygen species
SIRS systemic inflammatory response syndrome
SMA superior mesenteric artery
TNFα tumour necrosis factor alpha
XDH xanthine dehydrogenase
XOR xanthine oxidoreductase
5
LIST OF CHEMICAL COMPOUNDS
CH4 methane
CO carbon monoxide
CO2 carbon dioxide
H2 hydrogen
H2S hydrogen sulphide
NH3 ammonia
NO nitrogen monoxide
6
SUMMARY
In recent decades, cardiac surgical operations have become routine procedures, but the
risk of perioperative complications remains high. Pericardial tamponade (PT) is a
medical emergency leading to hypoperfusion of sensitive organs and, if untreated,
cardiogenic shock with circulatory collapse. The routine use of extracorporeal
circulation (ECC) in cardiac surgical interventions requiring cardiopulmonary bypass
(CPB) by itself poses a non-negligible risk of systemic inflammatory response and
organ damage.
This thesis focusses on the design of clinically relevant large animal models of
PT and CPB and the therapeutic application of methane (CH4) in PT- and CPB-induced
splanchnic and renal changes, especially on the modulation of inflammatory pathways.
We designed and conducted two series of experimental studies to investigate whether
CH4 has an effect on sensitive organs in PT-induced cardiogenic shock or CPB-induced
systemic inflammatory response.
In Study I, we performed experiments on three groups of minipigs. Group 1
served as the sham-operated control; in Groups 2 and 3, surgical PT was induced for
60 min with a novel technique with trans-phrenic pericardial cannula insertion. Group
3 was treated with 2.5% v/v normoxic CH4 through the ventilator. Haemodynamics and
mesenteric macro- and microcirculation were monitored, the components of mucosal
inflammatory response, intestinal mast cell activation and leukocyte function were
detected in tissue samples, and superoxide content was measured in whole blood
samples. Inhalation of CH4 had no impact on the macrohaemodynamic status of the
animals after the release of PT, but mucosal microcirculation and in vivo histology data
showed significant improvement. In line with these findings, both leukocyte
accumulation and mast cell degranulation decreased, and the superoxide amount of
whole blood was lowered in the CH4-treated group.
In Study II, we designed and employed an experimental model of CPB using
anaesthetized minipigs in two groups. Groups 1 and 2 both underwent 60 minutes of
centrally cannulated CPB. Group 2 received 2.5% v/v normoxic CH4 added to the
oxygenator sweep gas. Renal artery (RA) flow was monitored, and hourly diuresis was
measured. Activities of xanthine oxidoreductase (XOR) and myeloperoxidase (MPO)
were determined from cardiac, intestinal and renal tissue samples. Superoxide content
was measured in whole blood samples. CH4 administration improved the severe
impairment of RA flow and maintained the hourly diuresis in the recovery period after
ECC. XOR, a significant source of superoxide, was present with significantly lower
7
activity in cardiac, small intestinal and renal tissue samples. In line with these findings,
lower superoxide levels and reduced intestinal MPO activities were found, and the
inotropic demand was also lower in CH4-treated animals.
Overall, the evidence suggests that the application of CH4 in these porcine
models of cardiac surgery can effectively reduce organ damage by influencing XOR-
dependent ROS production, and leukocyte and mast cell activation. These results
suggest that exogenous CH4 may be a novel, additional therapeutic option to reduce the
risk of perioperative cardiac surgical inflammatory reactions.
8
TABLE OF CONTENTS
LIST OF PUBLICATIONS ........................................................................................... 2LIST OF ABBREVIATIONS ........................................................................................ 4LIST OF CHEMICAL COMPOUNDS ......................................................................... 5SUMMARY ................................................................................................................... 61. INTRODUCTION ................................................................................................... 101.1.Biologicalgasesandgasotransmitters.....................................................................................101.2.Methane.................................................................................................................................................101.3.Abioticandbioticmethanegeneration....................................................................................101.4.BioactivityofexogenousCH4–anti-inflammatoryeffects..............................................111.5.Theinflammatoryconsequencesoflowcardiacoutputstates.....................................121.6.Pericardialtamponade....................................................................................................................131.7.ECC-inducedsystemicinflammationincardiacsurgery..................................................131.8.Hypoperfusion-inducedintestinaldamage............................................................................14
1.8.1. Mast cell-associated mucosal injury ...................................................................... 141.8.2. Leukocyte recruitment and ROS production ......................................................... 16
1.9.Hypoperfusion-inducedkidneydamage.................................................................................171.10.InvivomodelsofPT-andCBP-inducedSIRS......................................................................17
1.10.1. Animal models of PT ........................................................................................... 171.10.2. Animal models of extracorporeal circulation ...................................................... 18
2. MAIN GOALS......................................................................................................... 193. MATERIALS AND METHODS ............................................................................. 203.1.Experimentalanimals......................................................................................................................203.2.Animalsandanaesthesia................................................................................................................203.3.Surgicalinterventions,StudyI(Aims1and3).....................................................................203.4.Surgicalinterventions,StudyII(Aims2and4)...................................................................223.5.Measurements....................................................................................................................................24
3.5.1. Haemodynamic measurements .............................................................................. 243.5.2. Measurement of blood CH4 content ....................................................................... 243.5.3. Tissue harvesting and processing .......................................................................... 243.5.4. Histology for light microscopy of leukocytes and mast cells ................................ 243.5.5. Tissue MPO activity .............................................................................................. 253.5.6. XOR activity .......................................................................................................... 253.5.7. Whole blood superoxide production ...................................................................... 253.5.8. Microcirculatory measurements ............................................................................ 263.5.9. In vivo detection of mucosal damage ..................................................................... 26
3.6.Experimentalprotocols..................................................................................................................273.6.1. Experimental protocol in Study I ........................................................................... 273.6.2. Experimental protocol in Study II ......................................................................... 27
3.7.Statisticalanalysis.............................................................................................................................284. RESULTS ................................................................................................................ 284.1.StudyI.....................................................................................................................................................28
4.1.1. Changes in haemodynamic parameters .................................................................. 284.1.2. Changes in microcirculation .................................................................................. 314.1.3. Intestinal leukocyte accumulation ......................................................................... 324.1.4. Mucosal mast cell degranulation ........................................................................... 32
9
4.1.5. In vivo histology ..................................................................................................... 334.1.6. Whole blood superoxide content ........................................................................... 35
4.2.ResultsofStudyII.............................................................................................................................364.2.1. Changes in renal arterial blood flow and hourly diuresis ...................................... 364.2.2. Changes in blood CH4 level ................................................................................... 374.2.3. Changes in MPO activity ....................................................................................... 374.2.4. Changes in XOR activity ....................................................................................... 39
5. DISCUSSION .......................................................................................................... 425.1.StudyI.....................................................................................................................................................425.2.StudyII...................................................................................................................................................445.3.Limitations...........................................................................................................................................46
6. SUMMARY OF NEW FINDINGS ......................................................................... 487. REFERENCES ........................................................................................................ 498. ACKNOWLEDGEMENTS ..................................................................................... 569. ANNEX .................................................................................................................... 57
10
1. INTRODUCTION
1.1. Biological gases and gasotransmitters
Many gases in the Earth’s atmosphere, such as oxygen (O2) and carbon dioxide
(CO2), are physiologically important or biologically irreplaceable. A number of other
gases that occur naturally, such as nitric oxide (NO), carbon monoxide (CO) and
hydrogen sulphide (H2S), are considered “gasotransmitters”, with diverse effects on the
basal functions of aerobic cells. According to current views, a member of this category
of gas must adhere to four characteristics (simplicity, availability, volatility and
effectiveness) and meet six criteria: (a) small molecules, (b) freely permeable to
membranes, (c) endogenously generated in mammalian cells with specific substrates
and enzymes, (d) serve well-defined specific functions at physiologically relevant
concentrations, (e) functions can be mimicked by their exogenously applied
counterparts, and (f) have specific cellular and molecular targets (1). In general,
gasotransmitters play vital regulatory roles in maintaining homeostasis in the human
body through distinct biochemical pathways.
1.2. Methane
Methane (CH4) is also part of the gaseous environment which maintains the
aerobic metabolism, but its role in cellular physiology is largely unmapped, and
relevant data on human cardiovascular effects is sparse. It is an inert, colourless,
odourless organic molecule, the main component of natural gas, with low solubility in
water (0.02 g/kg of water at 22°C room temperature). It is a simple asphyxiant, which
means that tissue hypoxia might occur when an increasing concentration of CH4
displaces inhaled air in a restricted area. In the atmosphere – which currently contains
approximately 1.7–2.0 ppmv (parts per million by volume) – CH4 is an important
greenhouse gas.
1.3. Abiotic and biotic methane generation
There are two main forms of methanogenesis, abiotic and biotic generation.
Abiotic CH4 formation is mainly associated with emissions from mining and
combustion of fossil fuels, and the burning of biomass, where CH4 formation is possible
at high temperatures and/or elevated pressures, which are irrelevant in biological
environments (2). Biotic CH4 formation takes place through anaerobic fermentation by
a unique class of prokaryotes (archaea) in ruminants or other mammals or in the
environment, where organic matter decomposes in the absence of oxygen or other
11
oxidants. The predominant methanogenic archaeon in the human large intestine is
Methanobrevibacter smithii (3), and the catalyzing enzyme of this pathway is methyl
co-enzyme M reductase, with the use of CO2 and H2 as main substrates (1).
In contrast to ruminants, which all emit substantial amounts of CH4, only around
one third of humans are considered to be CH4 producers. In humans, CH4 producers
and CH4 non-producers are distinguished clinically by breath analysis, where
production is defined as an increase of more than 1 ppm above the atmospheric level in
the exhaled air (4). However, it has been shown recently by precise measurement
techniques that CH4 production can be detected in all individuals (5), although in quite
a wide range of magnitude and dynamics; therefore, there seems to be a discrepancy
between older data and the latest. In addition, there is an ongoing debate whether CH4
production is genetically or environmentally determined or whether it is a result of yet
unknown factors. Indeed, other studies have confirmed direct CH4 release from
eukaryotes, including plants and animals, in the absence of microbes and in the
presence of oxygen (6–8). Further, several data have established the possibility of biotic,
non-archaeal CH4 generation in eukaryotic cells under various conditions involving
mitochondrial dysfunction (4). In summary, the overall evidence suggests that the
excretion of CH4 in the breath of mammals reflects not only intestinal microbial
fermentation, but also unidentified generation from target cells (9).
1.4. Bioactivity of exogenous CH4 – anti-inflammatory effects
The current evidence does not fully support the gasotransmitter concept, but it
does support the notion that CH4 is bioactive. Although CH4 is widely regarded as
inactive, a number of studies have demonstrated explicit biological effects in living
biological systems (10). An anti-inflammatory potential for CH4 was first reported in a
canine model of intestinal ischaemia-reperfusion (I-R) (11), and thereafter other reports
have confirmed the antioxidant and anti-apoptotic effects of exogenous CH4
administration in various in vitro and in vivo settings (12–21). The exact mechanism of
action is not fully clarified, but it has been shown that the anti-inflammatory action is
mediated through the inhibition of polymorphonuclear (PMN) leukocyte activation and
reduced reactive oxygen species (ROS) production (11). Other experimental data have
also shown that the biological effects are related to the inhibition of xanthine
oxidoreductase (XOR) activity as well, thus lowering the amount of superoxide radical
formation (22). Superoxide is one of the most important reactive oxygen species (ROS)
and a potent activator of effector cells like polymorphonuclear (PMN) leukocytes and
12
mast cells in the small intestine. In this line, a previous study has demonstrated that
exogenous CH4 inhalation was protective in I-R-induced nitrergic neuronal changes
and inhibited XOR-linked reactions during hypoxia-mediated intestinal injury (22).
Other findings provided evidence that administration of CH4-enriched saline solutions
is protective in experimental liver failure (12), autoimmune hepatitis (23), acute lung
injury (15), and hepatic (13), spinal cord (15), retinal (16), myocardial (17) and skin I-
R injuries (18). Today the overall evidence points to the likelihood that exogenous CH4
can influence different intracellular signalling pathways, which may be therapeutically
exploited to attenuate the signs and consequences of inflammation (24).
1.5. The inflammatory consequences of low cardiac output states
Perioperative local and/or systemic inflammation due to the natural aetiology
of cardiac diseases and/or the invasive nature of technical interventions represents a
major correlative risk of cardiac surgery and a key component of postoperative
complications. Maintaining cardiac function is essential in keeping the distal perfusion
of organs before, throughout and after the surgical procedure. Low cardiac output (CO)
states, such as pericardial tamponade (PT), post-cardiotomy cardiac stunning and
perioperative myocardial infarction, often lead to cardiogenic shock, with a high risk
of morbidity and mortality. Nevertheless, permanently low CO, even without definite
signs of cardiogenic shock, can induce microcirculatory impairment in peripheral
tissues, triggering local and later systemic inflammatory responses. In this respect, early
measures of management are clearly needed to avoid permanent organ failure or death
(25).
Furthermore, reperfusion, i.e. the restoration of blood flow after a period of
ischaemia, can also place previously ischaemic organs at risk of further cellular necrosis
and limited recovery (26). This phenomenon is termed I-R injury. Reperfusion injury
is mainly mediated by ROS formation in the re-oxygenized tissues, and the main source
in the intestines is xanthine oxidoreductase (XOR) and accumulated PMNs, a second
wave cellular response of critical importance in mucosal I-R damage. The process is
often associated with microvascular injury and the dysfunction of the endothelium,
leading to increased permeability of capillaries, damage to plasma membranes, proteins
and DNA in the cells (27).
13
1.6. Pericardial tamponade
Among the major complications of cardiac surgical interventions, PT is
considered as one of the most dramatic pathological conditions affecting both the
macro- and microhaemodynamics of the human body. The lack of urgent treatment can
lead to cardiogenic shock and is inevitably followed by multi-organ failure and death
(28). PT is caused by fluid accumulation within the pericardial sac. Transudation can
be a consequence of severe renal failure, neoplastic diseases or end-stage heart failure
(29). Inflammatory diseases with various aetiologies can lead to massive pericarditis
and purulent pericardial effusions. However, PT can most likely be an early or late
haemorrhagic complication of acute type-A aortic dissection, chest trauma, or cardiac
surgical procedure or intervention. The mortality rate is usually very high, but due to
the large aetiological variability, exact statistical data are rarely available; thus,
comparisons between different pathologies are pointless. In a high volume patient care
centre, the all-cause in-hospital mortality was shown to be approx. 20% and the 30-day
mortality ranged from 25 to 35% (30). The rate of fluid accumulation within the
pericardial sac determines the haemodynamic effects and the clinical picture as well.
The 2015 ESC clinical guidelines on the management of pericardial diseases propose
an echocardiographic (ECHO) classification based on the largest ECHO-free space and
step-by-step scoring system-based management (31). Nevertheless, understanding the
exact pathophysiologic mechanisms of PT is the key to taking the necessary diagnostic
steps and to choosing the optimal therapeutic options for patients.
Acute PT is a rapidly progressive condition, in which there is usually no time
for slower compensatory mechanisms to develop. It has been shown that strong
sympathetic neurohormonal activation evolves and that the release of endogenous
vasopressors is also significant (32). Peripheral vasoconstriction elevates the afterload,
which can worsen distal organ hypoperfusion and increases the myocardial oxygen
demand by elevating left ventricular workload. When the acute compensatory
responses are depleted, rapid circulatory collapse develops.
1.7. ECC-induced systemic inflammation in cardiac surgery
Since the first successful open-heart surgery with cardiopulmonary bypass
(CPB) by John Gibbon on May 6, 1953, thousands of such procedures have been carried
out worldwide each year. However, despite the safety of modern CPB circuits,
extracorporeal circulation (ECC) is still associated with some degree of post-operative
inflammatory activation, similar to the septic systemic inflammatory response (SIRS).
14
It is generally triggered immediately during CPB, and if the circulatory support is
prolonged, e.g. during a complex operation requiring deep hypothermic circulatory
arrest, the degree of inflammatory response can be much higher with higher risk for
multi-system organ failures. Indeed, besides cardiogenic shock-induced I-R damage,
the ECC or other means of ECC, such as extracorporeal membrane oxygenation
(ECMO), can themselves contribute significantly to the evolution of SIRS after cardiac
surgery (33,34).
It has been shown that blood contact with the foreign surface of the CPB circuit
activates humoral and cellular factors (34), leading to the intrinsic activation of the
coagulation cascade. Due to the routine use of heparin before initiation of ECC,
thrombin and fibrin complexes are not formed, but the first steps of the coagulation
cascade are activated along with other pro-inflammatory cascades, such as the kinin–
kallikrein system or the complement system, triggering a wide variety of cellular
systems, such as endothelial cells, leukocytes, thrombocytes and mast cells (33). If
these cascade activations are dysregulated due to prolonged CPB time or metabolic
changes, significant tissue and organ damage can occur in sensitive organs such as the
kidneys and intestines. Therefore, a search for novel therapeutic strategies to reduce
post-CPB inflammatory damage is an important clinical and scientific research goal.
1.8. Hypoperfusion-induced intestinal damage
Among the organs affected by cardiogenic shock, the gut plays an important
role in preserving a solid barrier between the bloodstream and the outside world. It is
of vital importance to preserve the endothelial and epithelial functions of the mucosa
to prevent bacterial translocation and thus sepsis. Low CO states like PT have a strong
impact on mesenteric circulation by triggering local splanchnic hypoxia due to
endogenous vasoconstriction. A diverse system of cascades, including pro-
inflammatory cytokines and complement factors, enhances mucosal leukocyte
recruitment and amplifies the local inflammatory response. Among the numerous
vasoconstrictors, the endothelin-1 (ET-1) system plays a pronounced role in the
maintenance of splanchnic hypoxia, mucosal leukocyte accumulation and triggering
mast cell degranulation as well (35).
1.8.1. Mast cell-associated mucosal injury
The mucosal damage induced by low CO states is largely dependent on local
vasoactive responses which influence various secondary cellular mechanisms. Mast
15
cells are key participants in these responses (36)(37). These multifunctional cell
populations play various roles in the homeostatic stability of the connective tissue, and
are key elements in local vasoregulation. Mast cells are oval-shaped 4–24 μm cells,
morphologically similar to basophil leukocytes with about 40% of their cytoplasm
containing secretory granules (38). The main sub-populations can be classified
according to the anatomical position or the biochemical patterns defined by the granule
composition. Traditionally, Mucosal Mast Cells (MMCs) and Connective Tissue Mast
Cells (CTMCs) are distinguished based on their histochemical staining properties (39).
Both populations are present around capillaries or neurons, mainly in anatomical
locations which are close to barriers to the surrounding world, such as the skin, the
lungs and the mucosa in the digestive system. MMCs are mainly present in the sub-
mucosal layer in close proximity to postcapillary venules in the stomach, and small and
large intestines, and contain tryptase, a serine protease as active secretory ingredient.
CTMCs are mainly present in the connective tissue in the skin and contain chymase,
tryptase and carboxypeptidases (40). According to their anatomic position, MMCs can
respond to stimuli and outside effects induced by antigens, bacteria, viruses and fungi
by degranulation of preformed cytoplasmic vesicles, thus influencing the vasoactive
responses (41). In addition, mast cells can also generate and actively secrete de novo
formed mediators (cytokines and growth factors) and mediate the mobilization of other
inflammatory effector cells.
Among the biogenic mediators, MMCs contain histamine, serotonin and
dopamine; degranulation thus leads to vasodilation and oedema formation through
increased capillary permeability (32-34). Preformed cytokines like interleukin-4 (Il-4),
Il-5, tumour necrosis factor (TNF) and chemokines can bring about leukocyte adhesion
and extravasation, thus augmenting the immune response. In the case of ongoing
inflammation, both leukocytes and MMCs will mediate and amplify tissue injury with
the release of proteases, such as chymase, tryptase, matrix metalloproteinases (MMPs),
and ROS formation (40,43–45).
The activation and degranulation of MMCs can proceed through two main
pathways. The immune pathway is immunoglobulin E (IgE)-mediated and high-affinity
IgE receptor (FcεRI)-linked and is a possible contributor to intestinal allergic diseases
(41). There is conflicting data on the non-immunologic activation pathways, but it is
likely that G protein-coupled receptor activation and intracellular pathways triggered
by MMC-produced substances play a vital role in I-R-derived MMC activation (46,47).
16
These involve endothelin (35), complement (48) and adenosine receptor (49)-linked
activation. It is also known that ROS is an important component of the biochemical
pathway in MMC degranulation, and in fact inhibition of ROS formation can prevent
degranulation (45).
1.8.2. Leukocyte recruitment and ROS production
To date much progress has been made in understanding the different
biochemical pathways which lead to I-R damage (26). Leukocyte recruitment after
intestinal I-R is a multi-pathway process with the involvement of cellular interactions
and chemotactic signals. The primary site of leukocyte migration is across the
endothelial wall of post-capillary venules. Each endothelial tissue has a specific pattern
of anchoring transmembrane glycoproteins, such as P- and E-selectins from the selectin
family, α4- and β2-integrins, and intercellular adhesion molecule 1 (ICAM-1) for
leukocyte capturing, binding and activation (44). The dynamics of inflammation are
thus unique for every tissue. This is also true for the expression of chemotactic patterns
(IL-1,-3,-6,-8 and TNF) (50)(38) by the cells involved in immunomodulation, most
importantly mast cells, endothelial cells and leukocytes.
Reperfusion-mediated ROS production is a significant contributor to ischaemic
mucosal damage. ROS formation can occur in the mitochondria throughout the hypoxic
phase and after reperfusion as well, but the key ROS producer is most probably
intestinal XOR (51), which is present in high concentration in many species, including
humans. In normoxic states, xanthine dehydrogenase (XDH - EC 1.17.1.4) is a key
enzyme in purine catabolism, which converts xanthine to urate with nicotinamide
adenine dinucleotide (NAD+) and H2O as a proton acceptor (52). In hypoxic conditions,
however, the enzyme is reversibly or irreversibly converted to xanthine oxidase (XO)
form (EC 1.17.3.2). During reperfusion, XO will catalyse the conversion of 1 mol
hypoxanthine to xanthine and to uric acid formation with the production of 2 mol
superoxide (O2.-), which will act as a substrate for other ROS and nitrosative radical
species formation, a key factor in tissue leukocyte recruitment (53).
17
1.9. Hypoperfusion-induced kidney damage
CPB often affects the renal function of patients undergoing cardiac surgery, and
acute kidney injury (AKI) is one of the most frequent complications of these
procedures. The pathogenesis of the syndrome is complex and influenced by many
factors, but it has been established that perioperative low CO or hypoperfusion in
association with cardiogenic shock or hypovolemia can play critical pathogenic roles
in the development and progression of AKI. Nevertheless, the exact molecular
pathomechanism is still unclear, and it has been suggested that further research should
be focussed on the investigation of novel supportive therapies and the development of
new pharmacological approaches aimed at reducing AKI occurrence. In this line, there
is evidence that the activation of the ET-1 system may be a potent mediator of
vasoconstriction, inflammation and oxidative stress reaction that could lead to AKI. It
has been demonstrated that the administration of ET-A receptor antagonist compound
reduced the number of inflammatory cells and improved kidney function as well in a
pig model of post-CPB kidney injury (54).
1.10. In vivo models of PT- and CBP-induced SIRS
1.10.1. Animal models of PT
In a seminal paper, Lluch et al. described a canine model of cardiogenic shock,
where the left ventricular function was reduced by coronary embolization (55). Since
then, numerous refinements have been made to study the immediate and late macro-
and microhaemodynamic consequences, but a reproducible, well-controlled and stable
experimental setting is still needed. The in vivo reduction of myocardial function can
be carried out relatively easily by selective coronary ligation or embolization, but the
technique is accompanied by high mortality due to malignant arrhythmias. On the other
hand, it is possible to reduce cardiac output by obstructing ventricular filling and
lowering the left ventricular stroke volume, a condition that can be achieved by
inducing PT. Our research group has previously demonstrated that PT is indeed an
appropriate model to study the pathomechanism of cardiogenic shock (32). These
experiments were carried out on anaesthetized, mechanically ventilated dogs, and it has
been shown that peripheral haemodynamics and vasoactive humoral responses could
be monitored in sufficient detail (32). In this setup, PT is induced by infusion of saline
through a cannula placed in the pericardium via thoracotomy, and the progression could
be immediately terminated by fluid extraction from the pericardium. This has thus
become a standard model for studying the consequences of reversible cardiogenic
18
shock (56). The inherent disadvantage of the model is thoracotomy itself and the
possibility of impaired lung function.
1.10.2. Animal models of extracorporeal circulation
The technological development of human ECC devices capable of providing
sufficient perfusion was based on a variety of in vivo experiments and animal trials.
Since then, many refinements have been made to perfect the CBP technique for clinical
use; however, basic problems, such as inflammatory reactions and cellular damage to
circulating blood, have yet to be solved. Besides the technological needs of ECC circuit
development, the basic pathophysiological problems require the use of well-established,
stable animal models. Due to the high costs of CBP components and supplemental
expenses, in vivo ECC studies are performed in only few research institutions. Swine,
dogs, cows and sheep are usually employed as models (57), but the general
disadvantage of heavy equipment use and the requirement of a full-scale operative
environment remain. It would be possible to perform small animal experiments, using
rodents (58), to reduce costs, time and the need for manpower, but there are many other
disadvantages, including species differences and the operative challenges in such small
scales. In this sense, the porcine model of CPB is still considered the best platform for
the translational development of novel organ protection strategies.
2. MAIN GOALS
Cardiac surgery-related perioperative inflammatory activation is a significant
clinical problem, and there is a clear need for efficient adjuvant therapies to influence
the outcome of such events. Our first aim was to develop and design suitable in vivo
models, where the characteristics of the postoperative clinical syndrome can be
examined under standardized conditions.
1. A main disadvantage of the currently used large animal PT models is thoracotomy
itself with impaired lung function and extended wound surface with a high risk of
bleeding complication, and lung and tissue injury. Our aim was to design an
experimental procedure to model iatrogenic PT, where thoracotomy can be avoided,
but the situation is realistic, similar to the clinical appearance.
2. Secondly, we aimed to characterize the ECC-induced haemodynamic and
microcirculatory consequences in a clinically relevant larger animal model with
anatomical and immunological similarities to humans.
In these experimental protocols, we placed special emphasis on identifying the
characteristic haemodynamic and microcirculatory changes and main components of
the peripheral inflammatory response, including signs of mucosal mast cell and
leukocyte activation. Thereafter, the main purpose of our succeeding studies was to
verify and characterize the effects of exogenous CH4 administration in these model
scenarios.
3. Previous evidence demonstrated the anti-inflammatory capacity of CH4 with the
possibility of modulating the pathways leading to oxidative stress. Therefore, our next
aim was to outline the intestinal effects of CH4 inhalation in a standardized large animal
model of experimental PT. For this purpose, normoxic CH4 was administered through
the ventilator of anaesthetized minipigs during PT-induced non-occlusive splanchnic I-
R.
4. We also aimed to investigate whether the systemic inflammatory response caused by
ECC could be modified with exogenous CH4. The additional aim of this study was to
determine whether CH4 administration would influence the development of post-CPB
kidney dysfunction. We investigated this hypothesis in anaesthetized minipigs with
centrally cannulated ECC with normoxic CH4 added to the oxygenator gas sweep.
20
3. MATERIALS AND METHODS
3.1. Experimental animals
The experiments were performed in accordance with National Institutes of
Health guidelines on the handling and care of experimental animals and EU Directive
2010/63 on the protection of animals used for scientific purposes. The studies were
approved by the Animal Welfare Committee of the University of Szeged (approval
number: V/148/2013).
3.2. Animals and anaesthesia
28 male outbred Vietnamese minipigs (weighing 45±8 kg) were used (n=18 in
Study I, n=10 in Study II). A mixture of ketamine (20 mg kg-1; CP-Ketamin 10%;
Pordulab Pharma-induced anaesthesia. Raamsdonksveer, Netherlands) and azaperone
(2 mg kg-1; Stresnil; Elanco Animal Health, Vienna, Austria) was administered
intramuscularly. After endotracheal intubation, mechanical ventilation was started with
a tidal volume of 10 ml kg-1, and the respiratory rate was adjusted to maintain the end-
tidal pressure of CO2 and partial pressure of CO2 in the 35–45 mmHg ranges.
Anaesthesia was maintained with a continuous infusion of propofol (50 μl min-1kg-1 iv;
6 mg kg-1h-1; Propofol; Fresenius Kabi, Bad Homburg, Germany), midazolam (1.2 mg
kg-1h-1; Midazolam Torrex; Torrex Chiesi Pharma, Vienna, Austria) and fentanyl (0.02
mg kg-1h-1; Fentanyl-Richter; Richter Gedeon, Budapest, Hungary), while muscle
relaxation was maintained with pipecuronium (single administration, 4 mg dose;
Arduan; Richter Gedeon, Budapest, Hungary).
3.3. Surgical interventions, Study I (Aims 1 and 3)
The left jugular vein was cannulated for fluid and drug administration. The left
femoral artery was cannulated for the measurement of mean arterial pressure (MAP),
heart rate (HR) and CO by transpulmonary thermodilution (PiCCO Catheters;
PULSION Medical Systems, Feldkirchen, Germany). MAP, HR and CO data were
recorded, while pressure signals (BPR-02 transducer; Experimetria Ltd, Budapest,
Hungary) and superior mesenteric artery (SMA) flow signals (T206 Animal Research
Flowmeter; Transonic Systems Inc., Ithaca, NY, USA) were measured continuously
and registered with a computerized data-acquisition system. Urine output was assessed
by surgically placing a urinary catheter in the bladder via the femoral incision. The
urine was collected and measured at the end of the observation period to calculate
average hourly diuresis. Ringer’s lactate was given at a rate of 10 mlkg-1h-1. After a
21
median laparotomy, a flow probe was placed around the SMA (Transonic Systems Inc.,
Ithaca, NY, USA). The wound in the abdominal wall was temporarily closed thereafter
with clips. In addition, intravital examination of intestinal microcirculation was carried
out with an orthogonal polarization spectral imaging system (OPS; Cytoscan A/R,
Cytometrics, Philadelphia, PA, USA), and the extent of damage to the gastric mucosa
was evaluated by in vivo histology (Five1, Optiscan Pty. Ltd., Melbourne, Victoria,
Australia). The diaphragm was accessed through a median laparotomy. A 3-cm incision
was made at the sternal part, avoiding the muscular region of the diaphragm. The
pericardium was opened, and a cannula was fixed into the pericardial cavity with a
pledgeted purse-string suture (Figure 1). The tamponade was induced and maintained
for 60 min by intrapericardial administration of heparinized own blood (100±50 ml),
and MAP was maintained at 40–45 mmHg. The abdominal wall was closed with
surgical clips during the observation period and between microcirculatory image
recordings.
Figure 1. The insertion of the pericardial cannula via the transphrenic route (1 diaphragm, 2 pericardium, 3 liver, arrow: the cannula fixed into the pericardium).
22
3.4. Surgical interventions, Study II (Aims 2 and 4)
The anaesthetized animals were placed in a supine position on a heating pad to
maintain body temperature between 36 and 37°C and received an infusion of Ringer’s
lactate solution at a rate of 10 ml kg-1 hr-1 during the experiments. The left jugular vein
was cannulated for fluid and drug administration, and the left femoral artery was also
cannulated to measure MAP, HR and CO by transpulmonary thermodilution. After a
median laparotomy, the renal artery (RA) was dissected free, and a flow probe was
placed around the blood vessel (Transonic Systems Inc., Ithaca, NY, USA) to measure
blood flow. The abdominal incision was temporarily closed with clips. A urinary
catheter was surgically placed in the bladder via the femoral incision. The urine was
collected and measured at the end of the observation period to calculate average hourly
diuresis.
A median sternotomy was performed and the pericardium dissected. The aorta
was dissected free and circled with tape. After the administration of heparin
(Heparibene; Teva, Budapest, Hungary) (2000 IU iv) and activated clotting time (ACT)
control (ACT > 500 sec), purse-string sutures were placed in the ascending aorta and
the right atrial appendage, and standard central CPB cannulation was performed
(Figures 2 and 3). Due to the lack of aortic cross clamping and the fragility of the upper
pulmonary vein, venting was not used. Cardiotomy suction was not employed because
the aorta or the cardiac chambers were not open. After the baseline haemodynamic
measurements, CPB was initiated and respiration stopped. CPB was carried out with a
modular perfusion system (Therumo Sarns 8000; Terumo Cardiovascular, Ann Arbor,
MI, USA) using a disposable hollow fibre oxygenator (CAPIOX®; Terumo
Cardiovascular, Ann Arbor, MI, USA). In addition, the cessation of the respiration
achieved maximal peak blood and cellular CH4 concentration in the treated animals.
The Du Bois formula was applied for non-invasive estimation of the CO to calculate
the body surface area, and 2.4 l/m2 CI was used standardly for all the animals. After the
insertion of the central venous line and the PiCCO cannula in the femoral artery, CO
was measured directly with the PiCCO system via thermodilution. The measured and
estimated CO values were nearly identical in all cases. Oxygenator gas sweeps were
adjusted to post-membrane arterial blood gas samples.
23
Figure 2. Schematic illustration of the surgical setting of CPB in Study II. Figure 3. The central cannulation of CPB in Study II.
3.5. Measurements
3.5.1. Haemodynamic measurements
MAP, CO and HR were monitored with the PiCCO Plus monitoring system
(PULSION Medical Systems; Munich, Germany). Blood flow signals (T206 Animal
Research Flowmeter; Transonic Systems Inc., Ithaca, NY, USA) were recorded and
registered with a computerized data acquisition system (SPELL Haemosys;
Experimetria, Budapest, Hungary).
3.5.2. Measurement of blood CH4 content
A near-infrared laser technique-based photoacoustic spectroscopy apparatus
(60) was employed to confirm the presence of exogenous CH4 in the blood. Blood
samples were taken from the aorta and jugular vein (5 ml from each) and placed in a
glass vial with 20 ml headspace volume and closed so as to be airtight. The outlet of
the vials was connected to the spectroscope pump, headspace gas was pumped into the
chamber of the device at a rate of 10 mL/min, and the CH4 content of the air above the
blood samples was measured. The CH4 values were corrected for background levels
and expressed in ppm.
3.5.3. Tissue harvesting and processing
Tissue biopsies kept on ice were homogenized in a phosphate buffer (pH 7.4)
containing 50 mM Tris-HCl (Reanal, Budapest, Hungary), 0.1 mM EDTA, 0.5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg ml-1 soybean trypsin
inhibitor and 10 μg ml-1 leupeptin (Sigma-Aldrich GmbH, Germany). The homogenate
was centrifuged at 4°C for 20 min at 24 000 g, and the supernatant was loaded into
centrifugal concentrator tubes (Amicon Centricon-100; 100 000 MW cut-off ultrafilter).
XOR activity was determined in the ultrafiltered supernatant, while that of MPO was
measured on the pellet of the homogenate.
3.5.4. Histology for light microscopy of leukocytes and mast cells
Full-thickness ileal biopsies taken at the end of the experiments were analysed
in each group. The tissue was fixed in 6% buffered formalin, embedded in paraffin, cut
into 4-μm-thick sections and stained with haematoxylin and eosin. The infiltration of
leukocytes was detected and the number of leukocytes counted in at least 20 fields of
view at an original magnification of 400x.
25
Intestinal biopsy samples for light microscopy were rapidly placed into ice-cold
Carnoy’s fixative and trimmed along the longitudinal axis. The fixed tissue was
attached to a hard cardboard backing to ensure the optimal longitudinal direction of the
section. The samples were embedded in paraffin, sectioned and stained with
haematoxylin-eosin, acidic toluidine blue (pH 0.5) or alcian blue-safranin O (pH 0.4).
MCs stained positively were quantitated in the villi of an average of 20 villus-crypt
units. The counting was performed in coded sections at 400 optical magnifications by
one investigator. Loss of intracellular granules and stained material dispersed diffusely
within the lamina propria were taken as evidence of mast cell degranulation.
3.5.5. Tissue MPO activity
Being a marker of tissue leukocyte infiltration, MPO activity was measured on
the pellet of the homogenate (61). Briefly, the pellet was resuspended in a K3PO4 buffer
(0.05 M; pH 6.0) containing 0.5% hexa-1,6-bis-decyltriethylammonium bromide. After
three repeated freeze-thaw procedures, the material was centrifuged at 4°C for 20 min
at 24 000 g, and the supernatant was used for MPO determination. Next, 0.15 ml of
3,3’,5,5’-tetramethylbenzidine (dissolved in DMSO; 1.6 mM) and 0.75 ml of hydrogen
peroxide (dissolved in K3PO4 buffer; 0.6 mM) were added to 0.1 ml of the sample. The
reaction led to the hydrogen peroxide-dependent oxidation of tetramethylbenzidine,
which could be detected spectrophotometrically at 450 nm (UV-1601
spectrophotometer; Shimadzu, Kyoto, Japan). MPO activities were measured at 37°C;
then the reaction was halted after 5 min with the addition of 0.2 ml of H2SO4 (2 M).
The data were expressed in terms of protein content.
3.5.6. XOR activity
XOR activity was determined in the ultrafiltered, concentrated supernatant with
a fluorometric kinetic assay based on the conversion of pterine to isoxanthopterine in
the presence (total XOR) or absence (XO activity) of the electron acceptor methylene
blue (62).
3.5.7. Whole blood superoxide production
The chemiluminometric method developed by Zimmermann et al. (63) was used
for the whole blood superoxide production measurements. During the measurements,
10 μl of whole blood was added to 1 ml of Hank’s solution (PAA Cell Culture,
Westborough, MA, USA), and the mixture was maintained at 37°C until assay. The
chemiluminometric response was measured with a Lumat LB9507 luminometer
26
(Berthold, Wildbad, Germany) during a 30 min period after the addition of 100 μl of
lucigenin.
3.5.8. Microcirculatory measurements
An intravital orthogonal polarization spectral imaging technique (Cytoscan
A/R; Cytometrics, Philadelphia, PA) was used for non-invasive visualization of the
mucosal microcirculation of ileum. This technique utilizes reflected polarized light at
the wavelength of the isosbestic point of oxy- and deoxyhaemoglobin (548 nm). As
polarization is preserved in reflection, only photons scattered from a depth of 200 to
300 μm contribute to image formation. A ×10 objective was placed onto the mucosal
surface of the small intestine, and microscopic images were recorded with a S-VHS
video recorder 1 (Panasonic AG-TL 700; Matsushita Electric, Osaka, Japan).
Quantitative assessment of the microcirculatory parameters was accomplished offline
with a frame-to-frame analysis of the videotaped images. Red blood cell velocity
(RBCV, μm/s) changes in the capillary were determined in three separate fields by
means of a computer-assisted image analysis system (IVM Pictron, Budapest,
Hungary). The same investigators performed all the microcirculatory evaluations.
3.5.9. In vivo detection of mucosal damage
The extent of damage to the ileal mucosa was evaluated by means of
fluorescence confocal laser scanning endomicroscopy (CLSEM) developed for in vivo
histology. Two investigators performed the analysis twice, separately. The mucosal
surface of the ileum was surgically exposed and laid flat for examination. The ileal
mucosal structure was recorded after the topical application of the fluorescent dye
acriflavine (Sigma-Aldrich Inc., St. Louis, MO, USA). The surplus dye was washed off
with saline 2 min before imaging. The objective of the device was placed onto the
mucosa, and confocal imaging was performed 5 min after dye administration (1
scan/image, 1024 x 512 pixels and 475 x 475 μm per image). The changes in the
mucosal architecture were examined with a semi-quantitative scoring system as
described previously (64). Briefly, (I) oedema (0 = no oedema, 1 = moderate epithelial
swelling, 2 = severe oedema) and (II) epithelial cell outlines (0 = normal, clearly, well-
defined outlines, 1 = blurred outlines, 2 = lack of normal cellular contours).
27
3.6. Experimental protocols
3.6.1. Experimental protocol in Study I
The animals were randomly allocated into three experimental groups (Figure 4).
Group 1 (n=6) served as a sham-operated control, with the same surgical interventions,
time frame and sampling as in Group 2 (n=6) but without the induction of PT. Group 3
received inhaled 2.5% v/v normoxic CH4 (Linde Gas, Hungary) in the last 5 minutes of
PT and for an additional 10 min after PT. In Groups 2 and 3, after the end of 60-min
PT, the blood was released from the pericardial sac and the animals were monitored for
180 min. Blood gases and haemodynamic parameters were measured every 30 min. An
in vivo histological examination on the ileal mucosa and detection of PMN leukocytes
were performed at baseline, 30 min after the relief of PT (90 min) and at the end of the
experiments (240 min). Tissue biopsies were harvested at the baseline, 30 min after the
relief of PT (90 min) and at the end of the experiments (240 min). Blood samples were
taken at the baseline 30 min after the relief of PT (90 min) and at the end of the
experiments (240 min).
Figure 4. The scheme of the experimental protocol for Study I.
3.6.2. Experimental protocol in Study II
The animals (n=10) were randomly allocated into two experimental groups (n=5,
each) (Figure 5). In both groups, CPB was maintained for 120 min and adjusted
throughout according to the CO calculated for the animals. The heart was not cross-
clamped nor arrested so as to exclude I-R and the accompanying inflammatory
activation. Left heart venting was not used because the aorta was not cross-clamped, so
the heart was left to beat and eject. After 120 min of CBP, flow was gradually decreased
and stopped. The cannulas were removed, and the cannulation points were surgically
closed. The non-treated CPB group (n=5) received a standard air–oxygen gas mixture
with 0.6 FiO2 at a flow rate of 2 l min-1 to the oxygenator. In the CH4-treated
28
(CBP+Met) group, 2.5% v/v normoxic CH4 (Linde Gas, Hungary) was added to the
oxygenator gas sweep at a rate of 1 l min-1. FiO2 values were adjusted to the post-
oxygenator PaO2 values. The CH4 content of the blood was monitored by near-infrared
laser technique-based photoacoustic spectroscopy after 60 min of CH4 treatment. The
duration of CPB and the post-CPB times did not differ between the groups. The post-
CPB haemodynamic stability (MAP > 60 mmHg) of the animal was maintained by
continuous iv volume replacement (Ringer’s lactate solution) and by maintaining the
systemic vascular resistance with perfusion of norepinephrine if necessary (0.05–0.35
μg kg-1 h-1; Arterenol; Sanofi-Aventis, Frankfurt am Main, Germany). After 120 min
of post-CPB monitoring, a blood sample was taken to detect whole blood superoxide
production, and small intestine, kidney and heart tissue biopsies were taken to measure
MPO and XOR enzyme activity.
Figure 5. The scheme of the experimental protocol for Study II.
3.7. Statistical analysis
Data analysis was performed with a statistical software package (SigmaStat for
Windows, Jandel Scientific, Erkrath, Germany). Friedman repeated measures analysis
of variance on ranks was applied within groups. Time-dependent differences from the
baseline for each group were assessed by Dunn’s method. The differences between
groups were analysed with the Mann–Whitney test. In the figures, median values and
75th and 25th percentiles are given; p values < 0.05 were considered significant.
4. RESULTS
4.1. Study I
4.1.1. Changes in haemodynamic parameters
The average duration of the surgical preparation phase was 70±20 min. MAP
remained at 40–45 mmHg as previously planned throughout the 60 min of PT,
accompanied by a concomitantly decreased CO and elevated HR (Table 1). After the
release of PT, MAP started to increase. However, by the end of the observation period,
29
it was significantly lower compared to the baseline values and to the MAP for the sham-
operated group. No significant difference was observed between the PT and PT + CH4-
treated groups. During the post-tamponade phase, the CO returned to the baseline
values and there was no significant difference between the three groups. The same
changes could be observed in the HR values (Table 1). The decreased venous return
was evidenced by a significant elevation of CVP throughout the PT phase (data not
shown). The SMA flow showed a significant drop in the PT phase as expected (Figure
6). After the release of PT, the SMA flow dramatically increased but did not reach the
baseline values. Differences in SMA flow between CH4-treated and non-treated groups
were not observed.
Group -5 min 60 min 120 min 180 min 240 min
Cardiac index (L/min-1 m-2)
Sham- operated
2.6 2.4; 2.7
2.4 2.3; 2.6
2.3 2.2; 2.5
2.4 2.2; 2.8
2.4 2.1; 2.7
Cardiac tamponade
2.5 2.2; 3
1.2*x 1.2; 1.4
2.2 1.9; 2.3
2.4 2.2; 2.8
2.1 2; 2.3
Cardiac tamponade +
methane
2.4 2.1; 2.9
1.1*x 1.1; 1.2
2 1.9; 2.7
2.2 2; 2.3
2.1 1.9; 2.2
Mean arterial pressure (mmHg)
Sham-operated
93.4 87.4; 104.3
101.6 89.1; 111.3
100.9 85.4; 112.1
93.3 83.4; 98.6
94.3 82.3; 101.5
Cardiac tamponade
95 91; 101
43.1*x 39.5; 44
75 68; 86
71*x 66.5; 77
72.5*x 63.5; 83
Cardiac tamponade +
methane
98 95.5; 104
46*x 45; 48.5
78.5 69; 111
77.5 x 66.5; 93
73*x 70; 80
Heart rate (beat min-1)
Sham-operated
83.5 71.1; 99.9
75.8 66.3; 85
82.5 67.8; 105.2
87.6 75.6; 104.5
94.6 76.1; 112
Cardiac tamponade
76.1 64.5; 112
118.8*x 108.5; 144.1
110.9 90.9; 120.5
115.8 85.1; 128
125.1* 102.1; 139.4
Cardiac tamponade +
methane
80.3 64; 105
114*x 108; 140.5
112.6* 97; 134.5
112.6* 98.5; 130
111 88.5; 124
Table 1. Changes in haemodynamics. Cardiac index (L/min-1 m-2), mean arterial pressure (mmHg) heart rate (beat min-1). *P<0.05 within group vs. baseline values (Friedman test followed by Dunn’s post-hoc test) and XP<0.05 vs. sham-operated group (Kruskal–Wallis test followed by Dunn’s post-hoc test).
31
Figure 6. Changes in superior mesenteric artery flow in the sham (n=6; white square-solid line), PT (n=6; grey circle-solid line) and PT+CH4 (n=6; white triangle-solid line) groups. 4.1.2. Changes in microcirculation
The RBCV did not change in the sham group, while a significant decrease from
the baseline was detected in the PT and PT + CH4-treated groups. At the end of the
observation period, the CH4-treated group showed a significant increase in RBCV
compared to the non-treated PT group (Figure 7).
Figure 7. Changes in red blood cell velocity in the sham (n=6; white box), PT (n=6; grey box) and PT+CH4 (n=6; diagonal line in grey box) groups.
32
4.1.3. Intestinal leukocyte accumulation
The leukocyte accumulation in the ileal tissue samples increased in the PT group
compared to the sham group. The CH4-treated group showed a significant decrease in
the leukocyte accumulation compared to the non-treated PT group (Figure 8).
Figure 8. Leukocyte content of the ileal biopsy in the sham (n=6; white box), PT (n=6; grey box) and PT+CH4 (n=6; diagonal line in grey box) groups.
4.1.4. Mucosal mast cell degranulation
The mast cell degranulation of the ileal tissue samples significantly increased
after 90 minutes in the PT group and remained significantly high at the end of the
observation period. This increase of degranulation was not observed in the CH4-treated
PT group at 90 minutes or at 240 minutes of the protocol (Figure 9).
33
Figure 9. Percentage of mast cell degranulation in the ileal biopsy in the sham (n=6; white box), PT (n=6; grey box) and PT+CH4 (n=6; diagonal line in grey box) groups.
4.1.5. In vivo histology
The mucosal morphology was examined with the intravital CLSEM technique
in real time. The epithelial morphology of the small intestinal mucosa presented normal
morphological patterns in the sham animals and in the baseline samples of the PT group
and CH4-treated group, while 30 min after PT induction longitudinal fissures appeared
and partial epithelium defects were detected in the non-treated PT group. The lack of
epithelium was extended in the end of observation. This extent of damage was not
observed in the CH4-treated PT group at 90 minutes or at 240 minutes (Figures 10AB).
The scores of the mucosal damage were significantly higher in both non-treated and
CH4-treated groups at 90 min and 240 min of the experiment. However, CH4 treatment
significantly decreased the score of mucosal damage compared to the non-treated PT
group (Figures 10AB).
34
Figure 10A. In vivo histology (CLSEM technique) showing the changes of the epithelial surface of the terminal ileum after acriflavine staining. The mucosal surface of the sham group (A, D, G), the structure of the mucosal surface in the PT group (B, E, H) and the structure of the mucosal surface in the PT+CH4 group (C, F, I) are shown.
Figure 10B. Changes in the values of scores of the in vivo histology in the sham (n=6; white box), PT (n=6; grey box) and PT+CH4 (n=6; diagonal line in grey box) groups.
35
4.1.6. Whole blood superoxide content
The superoxide content of the blood showed no difference in the baseline values
for the three groups. After 30 min of the release of PT, the amount of superoxide
significantly increased in the non-treated group compared to the sham group. In the
CH4-treated group, this increase was not observed. At 240 min significant differences
in superoxide content could not be shown between the groups (Figure 11).
Figure 11. Changes in whole blood superoxide production in the sham (n=6; white box), PT (n=6; grey box) and PT+CH4 (n=6; diagonal line in grey box) groups.
4.2. Results of Study II
4.2.1. Changes in renal arterial blood flow and hourly diuresis
A significantly decreased RA flow was detected during the CPB and post-CPB
periods. CH4 addition through the oxygenator resulted in significantly higher renal
blood flow during the CPB period in contrast to the non-treated group (Figure 12A).
The hourly diuresis during the observation period was significantly higher in the CH4-
treated group than in the animals without treatment (Figure 12B).
Figure 12. Changes in renal artery blood flow (A) in the ECC (n=5; black circle with continuous line) and ECC+Met (n=5; empty diamond with solid line) groups. Hourly diuresis (B) in the ECC (empty box) and ECC+Met (grey box) groups. The plots demonstrate the median (horizontal line in the box) and the 25th and 75th percentiles. # p < 0.05 between the CH4-treated and ECC groups (Mann–Whitney one-way analysis of variance on ranks).
4.2.2. Changes in blood CH4 level
The photoacoustic spectroscopy data showed a 1.4 ppm increase in CH4
concentration in the aortic blood sample above the background CH4 level. The CH4
concentration, measured from the samples from the jugular vein, was significantly
lower compared to the CH4 levels in the aortic samples (Figure 13).
Figure 13. CH4 content of the blood in the aorta (empty box) and jugular vein (grey box).
4.2.3. Changes in MPO activity
The rate of neutrophil accumulation in the intestinal, cardiac and renal tissue
was determined by measurement of MPO activity. Intestinal MPO activity was
significantly lower in the CPB–CH4 group in contrast to the non-treated group (Figure
14A). The MPO level in the cardiac and renal tissue showed no differences between
the two groups (Figures 14BC).
38
Figure 14. Changes in small intestinal (A), cardiac (B) and renal (C) MPO activity in the ECC (n=5; empty box) and ECC+Met (n=5; grey box) groups.
4.2.4. Changes in XOR activity
In the non-treated group, the XOR activity in all examined tissues was
significantly higher 2 h after the CPB period compared to the values for the group with
exogenous CH4 administration (Figures 15ABC).
Figure 15. Changes in small intestinal (A), cardiac (B) and renal (C) XOR activity in the ECC (n=5; empty box) and ECC+Met (n=5; grey box) groups.
40
4.2.5. Changes in blood superoxide production
Whole blood superoxide production was reduced by CH4 administration at the
end of the experiment in contrast with the control CPB group (Figure 16).
Figure 16. Changes in whole blood superoxide production in the ECC (n=5; empty box) and ECC+Met (n=5; grey box) groups.
4.2.6. Norepinephrine demand
During the post-CPB period, norepinephrine was administered to maintain a
minimum of 60 mmHg MAP. We found significant differences between the inotropic
demands of the 2 experimental groups. The CH4-treated group required significantly
less (p = 0.006) norepinephrine support compared to the non-treated group (Figure 17).
Figure 17. Changes in norepinephrine demand in the ECC (n=5; empty box) and ECC+Met (n=5; grey box) groups.
5. DISCUSSION
Cardiac surgery is a high-risk surgical category, where adverse circulatory events,
including deterioration of intra-abdominal perfusion, can lead to excess morbidity and
mortality. The main goal of this thesis was to model potentially harmful complications
and consequences of clinical practice and to test possibilities of risk reduction with a
novel, adjunctive treatment.
5.1. Study I
In the first protocol we investigated the overall characteristics and the mesenteric
consequences of temporary mechanical compression of the heart leading to
microcirculatory I-R of the splanchnic system. Experimental PT is a good model to
study the pathomechanism of cardiogenic shock (32,56,65,66). but it has several
limitations as well. The inherent disadvantage of the model is thoracotomy itself, the
possibility of impaired lung function and the risk of bleeding. We attempted to improve
the original model through several modifications. Most importantly, changing the
species to swine (according to accepted ethical and legal requirements and standards)
increased the translational power of the model. There are many anatomical similarities
between the human and porcine heart (e.g. the structure of the coronary artery system
and the lack of a collateral blood supply) and there are significant similarities, such as
the NOS pathway, which is also almost identical in pigs and humans (67). A next, major
forward step was the elimination of thoracotomy and the exclusive use of laparotomy
for intestinal monitoring and pericardial cannula insertion (68)(69). In our new model,
pledgets are placed in the pericardial purse-string suture line to seal the possible leaking
points. This technique minimizes surgical trauma and may contribute to the stability of
the experimental setting. Furthermore, we defined the importance of choosing the
proper type of fluid to fill the pericardial sac. In the case of saline, the risk of fluid
leakage and thus experimental instability is high; the technique was thus changed to the
most relevant colloid, heparinized own blood drawn from the central venous line. The
operative setup may not require special surgical skills, the tamponade can be easily
induced, and the release of PT can be easily executed. It may therefore be a well-
controllable model to investigate the inflammatory complications of central circulatory
disturbances. The model is stable and repeatable, and these qualities may make it a
good tool in the cardiovascular research arsenal.
The specific problem of low CO is often present in cardiac surgery, and there is
a high demand for possible new therapeutic options with a solution for the
43
inflammatory consequences. Earlier studies have demonstrated the potential beneficial
features of exogenous CH4 in several I-R models, with the delivery of CH4 by inhalation
or iv administration of CH4-enriched solutions (11)(12)(13)(17)(16). Therefore, we
investigated the effects of a normoxic air-CH4 mixture in a porcine model of PT-
induced I-R damage with special emphasis on mucosal inflammatory reactions. To our
knowledge, this translational approach has never been studied in vivo in the context of
cardiac surgery.
The induction of PT significantly influenced the macrohaemodynamics. MAP
and CO decreased, while CVP and HR rose. As a characteristic consequence of low
CO, the mesenteric circulation deteriorated and the dramatic decrease of the SMA flow
indicated the non-homogenous pattern of circulatory redistribution. The parallel in vivo
detection of the microcirculation provided evidence for the decreased intestinal
intramural perfusion. As expected, the haemodynamic disturbances were improved
after the relief of the PT, but MAP and CO did not return to the normal baseline level.
This circulatory state can be considered transitory, possibly returning to pre-PT values
after longer observation times.
We detected no differences after CH4 treatment in the macrohaemodynamics as
compared to the non-treated control group. However, the RBCV in the mucosa
significantly improved in the CH4-treated group 180 min after the release of PT. These
changes might be a consequence of the reduced pro-inflammatory activation, including
the degranulation of MMCs, which might have an influence on peripheral vascular
resistance.
Indeed, a higher proportion of activated mast cells were observed in the non-
treated PT group 30 min after the relief of PT, and the same histological picture was
seen after 240 min. In parallel, the accumulation of PMN leukocytes was also
significant as a direct consequence of I-R. As discussed before, there is evidence that,
in addition to IgE, non-immunologic G protein-coupled intracellular pathways play a
vital role in I-R-derived MMC activation (46,47). Thus, the depletion of secretory
granules and the release of various pro-inflammatory mediators could play a role in the
recruitment of leukocytes. In line with the microcirculatory changes at 30 min after the
release of PT, intravital CLSEM data also showed the first signs of structural damage
of the mucosa. In line with the reduction of the presence and activation of pro-
inflammatory cells, the extent of the structural damage to the mucosa was much lower
and the content of superoxide in the blood was also reduced by CH4 administration.
44
The available data on possible pathways of CH4 effects (i.e. anti-inflammatory,
antioxidant and anti-apoptotic) in various in vitro and in vivo experimental models were
recently reviewed (9). It has been suggested that many effects can be explained with
the physico-chemical properties of the non-polar gas. Until now no specific receptors
or enzymes have been found mediating the CH4 effects. However, there is evidence that
CH4 attenuates ROS production through the activation of antioxidant enzymes via the
Nrf2-ARE pathway (70). As a result of Nrf2-ARE, the activation of downstream
enzymes (i.e. haem oxygenase-1, catalase and superoxide dismutase) and lower
production of ROS is expected, leading to anti-inflammatory effects. In addition to
these intracellular signalling pathways, the mitochondrial electron transport system is
believed to be a potential target of CH4. Strifler et al. reported that leak mitochondrial
respiration improved in a medium containing 2.2% CH4–air mixture (71). These
potential effects of CH4 can be explained if CH4 dissolves in mitochondrial membranes.
It may thus influence membrane rigidity or change the conformity of trans-membrane
proteins in the respiratory chain, leading to alterations in ROS production. Therefore,
the reduction of the source of ROS, including a decreased level of leucocyte and mast
cell activation, may be associated with improved microcirculation and the overall better
structural status of the mucosal surface.
5.2. Study II
To date, many refinements have been made to improve the use of CPB in
cardiac surgery. Biocompatible circuits, the advanced oxygenator and pump design
(72) with shorter perfusion times have made postoperative patient recovery more
successful than ever before. Nevertheless, some degree of systemic inflammation with
an altered organ redox state still develops, and it is still not possible to overcome severe
inflammatory responses completely. Complications more often arise after longer
perfusion times or longer extracorporeal membrane oxygenation treatments. Blood
contact with foreign surfaces immediately triggers the intrinsic activation of the
coagulation cascade, leading to the broad activation of different pro-inflammatory
cascades and the PMNs, and the resultant oxidative stress limits postoperative recovery
(73)(74). The systemic inflammatory response syndrome was targeted by
corticosteroids, but a large-scale study showed no significant consequence in terms of
mortality and morbidity (75). Promising results have been achieved with cytokine
adsorption techniques in intraoperative cardiac surgery, but other data have shown no
reduction in the pro-inflammatory effect (76).
45
In our model, we present a novel method of delivering an effective anti-
inflammatory gas to improve the consequences of SIRS after CBP. The aim was to
simulate a centrally-cannulated extracorporeal bypass circuit for a relatively short
period of time. The cannulation of CPB and that of central VA-ECMO are in this case
nearly identical, but the air–blood interface in the venous reservoir, the use of an
occlusive roller pump, the duration of the extracorporeal circulation, the degree of
haemodilution and the lack of heparin-coated circuits rather resemble CPB. However,
based on the practical experience that can be derived from the model, we can say that
the induction of the inflammatory reaction due to extracorporeal circulation takes less
time and the reaction is more pronounced as compared to the use of a less damaging
ECMO circuit. We have shown that this technique is stable and repeatable and provides
a clinically relevant animal model to study the consequences of CPB-induced
inflammatory reactions. Renal function is frequently affected by CPB and there is
therefore a constant search for renal protection methods. In the present study, we
observed a significant decrease in renal arterial flow during and after CPB, and, despite
a decrease, the flow was significantly higher in the CH4-treated group than without the
addition of CH4. The increase also had a functional effect, as the hourly diuresis
remained in the low normal range in the CH4-treated group as compared to the oliguria
in the animals which had not received CH4 treatment.
According to findings reported by Vogel et al. (77), superoxide is capable of
directly increasing Ca2+ influx in the smooth muscle cells of renal afferent arterioles,
resulting in vasoconstriction, which may cause decreased renal blood flow. The
establishment of CPB inevitably leads to a certain degree of hypoperfusion and
activation of superoxide-producing enzymes such as NADPH-oxidase and XOR (73).
The significance of the effect of CH4 on oxidative stress enzymes is emphasized by the
lower superoxide level in the blood samples in the CH4-treated group. Another
important finding of our study is the decreased XOR activity in the cardiac tissue after
the addition of CH4. During tissue ischaemia and reperfusion, XOR is a significant
source of superoxide and a known contributor to oxidative stress. Allopurinol, a XOR
inhibitor, has a protective effect against I-R injury in different organs. This effect was
exploited in the case of the University of Wisconsin (UW) organ storage solution, the
gold standard in kidney preservation (78). In the present study, CH4 inhalation
significantly decreased renal XOR activity, thus possibly exerting an effect on renal
function similar to that of allopurinol. According to arterial and venous data,
approximately 2/3 of the administered exogenous CH4 was distributed across the body.
46
Since decreased XOR activity was detected in the cardiac and small intestinal samples
as well, this points to a systemic, non-organ-specific effect of CH4.
As discussed, the effects of CH4 are possibly due to the physico-chemical
properties of the gas (70)(71). Besides transport with the blood flow, the diffusion
through cell membranes can provide biologically accessible CH4 molecules, even in
less perfused areas (dissolved in lipids, non-polar regions of proteins etc.). In addition,
CH4 treatment also reduced MPO activity in the small intestine, suggesting decreased
leucocyte infiltration after CPB. Interestingly, this effect was absent in the heart and
kidney, a finding which is consistent with the experimental findings reported by
Murphy et al., who also noted the lack of PMN infiltration in the renal tissue 1.5 h after
CPB in a similar, relatively short-term, non-recovery set-up (79). We suggest that the
tissue-specific differences are due to the barrier nature and more pronounced immune
function of the intestinal mucosa. Here, the baseline MPO values are approx. 2
magnitudes higher as compared to those of other tissues. On the other hand, as a shock
organ, the GI tract is more exposed to the harmful effects of low flow and hypoxia
caused by redistributed circulation during and after CPB; therefore, inflammatory
activation may also be present on a much larger scale and in an earlier time frame. It
should be noted that the inotropic demand was also significantly lower in CH4-treated
animals. Norepinephrine can influence renal perfusion; however, the doses that were
administered in this study should not cause impairment of the renal circulation (80).
Moreover, Schaer et al. (81) reported that norepinephrine treatment increased renal
blood flow in dogs until the peak dose of 1.6 mg/kg/min, while the largest dose used in
the present study was 0.35 mg/kg/min. Further, the effect of CH4 treatment on renal
flow was already observed 60 min after the start of CPB, thus ruling out the possible
effect of inotropic treatment on this parameter.
5.3. Limitations
It is important to list the limitations of the studies. As pigs have immune
responses and cardiac anatomical and functional similarities comparable to humans
(82), in a study involving 4 species (i.e. rats, guinea pigs, swine and humans), the
activity of XOR in the pig heart was in the same range as that in human cardiac tissue,
and the response to allopurinol was also similar (83). Nonetheless, these animals were
healthy, without any accompanying cardiovascular alterations, while humans
undergoing cardiac surgical interventions and/or possible CPB use are usually in a
severely impaired cardiovascular condition prior to surgery. As beneficial as it is to
47
have an experimental PT model similar to humans, the use of a swine PT model consists
of numerous drawbacks due to the pathophysiologic characteristics of PT. PT is often
a hyper-acute state and is developed due to an existing cardiac pathology (aortic
dissection, cardiac surgery, intervention). It is therefore not possible to re-enact the
influences of pre-existing cardiac pathology on animal models. Also, multiple-series,
multiple-group experiments are difficult to carry out due to temporal and financial
limitations. In addition, technical limitations were also present in the models. The CPB
circuits used in cardiac operations have more complex tubing and use multiple roller
pump modules for venting, cardiotomy suction and cardioplegia delivery. Moreover,
cardiotomy suction aspirates blood from the surgical site, which has already been in
contact with tissue factor; thus, significant cellular activation has already taken place.
As the model lacked cardioplegia or any cardiac arrest, ischaemia of the cardiac tissue
was not present, thus possibly influencing the effectiveness of the treatment. However,
we provided evidence that CH4 administration might improve the consequences of PT
and CPB even under such circumstances.
48
6. SUMMARY OF NEW FINDINGS
1. We significantly refined a previous experimental technique and designed a new
animal model, which resembles iatrogenic PT. For this purpose, the pericardium
was accessed through the diaphragm via median laparotomy, and a cannula was
fixed into the pericardial cavity with a pledgeted purse-string suture.
Tamponade was induced and maintained by intrapericardial administration of
heparinized own blood.
2. We designed a simplified, but clinically relevant large animal model of CPB-
induced SIRS, where the haemodynamic and microcirculatory consequences of
ECC can be studied in sufficient detail.
3. We confirmed the bioactivity of CH4 in a large animal model of PT. The
decreased ileal leukocyte infiltration and mast cell degranulation with the
parallel reduction of blood superoxide concentrations demonstrate that
exogenous CH4 efficiently modulates the pathways of oxidative stress leading
to reduced structural damage of the mucosa.
4. We demonstrated for the first time that CH4 treatment is capable of increasing
renal blood flow and hourly diuresis in the post-CBP period. We also provided
evidence that the transmembrane diffusion of CH4 using a commercially
available hollow fibre membrane oxygenator is able to reduce the inflammatory
activation (neutrophil accumulation and XOR activity) in peripheral tissues (i.e.
heart, kidney and ileum) and the circulation as well (as evidenced by reduced
blood superoxide content), which indicates that systemic CH4 administration
may be a feasible way to modulate ECC-induced ROS production.
49
7. REFERENCES
1. Wang R. Gasotransmitters: growing pains and joys. Trends Biochem Sci. 2014 May;39(5):227–32.
2. Crutzen PJ, Andreae MO. Biomass burning in the tropics: impact on atmospheric chemistry and biogeochemical cycles. Science. 1990 Dec 21;250(4988):1669–78.
3. Eckburg PB. Diversity of the Human Intestinal Microbial Flora. Science. 2005 Jun 10;308(5728):1635–8.
4. de Lacy Costello BPJ, Ledochowski M, Ratcliffe NM. The importance of methane breath testing: a review. J Breath Res. 2013 Mar 8;7(2):024001.
5. Keppler F, Schiller A, Ehehalt R, Greule M, Hartmann J, Polag D. Stable isotope and high precision concentration measurements confirm that all humans produce and exhale methane. J Breath Res. 2016 Jan 29;10(1):016003.
6. Keppler F, Hamilton JTG, McRoberts WC, Vigano I, Braß M, Röckmann T. Methoxyl groups of plant pectin as a precursor of atmospheric methane: evidence from deuterium labelling studies. New Phytol. 2008 Jun;178(4):808–14.
7. Lenhart K, Bunge M, Ratering S, Neu TR, Schüttmann I, Greule M, et al. Evidence for methane production by saprotrophic fungi. Nat Commun. 2012 Jan;3(1).
8. Althoff F, Jugold A, Keppler F. Methane formation by oxidation of ascorbic acid using iron minerals and hydrogen peroxide. Chemosphere. 2010 Jun;80(3):286–92.
9. Mészáros AT, Szilágyi ÁL, Juhász L, Tuboly E, Érces D, Varga G, et al. Mitochondria As Sources and Targets of Methane. Front Med. 2017 Nov 13;4.
10. Pimentel M, Lin HC, Enayati P, van den Burg B, Lee H-R, Chen JH, et al. Methane, a gas produced by enteric bacteria, slows intestinal transit and augments small intestinal contractile activity. Am J Physiol-Gastrointest Liver Physiol. 2006 Jun;290(6):G1089–95.
11. Boros M, Ghyczy M, Érces D, Varga G, Tőkés T, Kupai K, et al. The anti-inflammatory effects of methane*: Crit Care Med. 2012 Apr;40(4):1269–78.
12. Yao Y, Wang L, Jin P, Li N, Meng Y, Wang C, et al. Methane alleviates carbon tetrachloride induced liver injury in mice: anti-inflammatory action demonstrated by increased PI3K/Akt/GSK-3β-mediated IL-10 expression. J Mol Histol. 2017 Aug;48(4):301–10.
13. Ye Z, Chen O, Zhang R, Nakao A, Fan D, Zhang T, et al. Methane Attenuates Hepatic Ischemia/Reperfusion Injury in Rats Through Antiapoptotic, Anti-Inflammatory, and Antioxidative Actions: Shock. 2015 Aug;44(2):181–7.
14. Sun A, Wang W, Ye X, Wang Y, Yang X, Ye Z, et al. Protective Effects of Methane-Rich Saline on Rats with Lipopolysaccharide-Induced Acute Lung Injury. Oxid Med Cell Longev. 2017;2017:1–12.
50
15. Wang W, Huang X, Li J, Sun A, Yu J, Xie N, et al. Methane Suppresses Microglial Activation Related to Oxidative, Inflammatory, and Apoptotic Injury during Spinal Cord Injury in Rats. Oxid Med Cell Longev. 2017;2017:1–11.
16. Liu L, Sun Q, Wang R, Chen Z, Wu J, Xia F, et al. Methane attenuates retinal ischemia/reperfusion injury via anti-oxidative and anti-apoptotic pathways. Brain Res. 2016 Sep;1646:327–33.
17. Chen O, Ye Z, Cao Z, Manaenko A, Ning K, Zhai X, et al. Methane attenuates myocardial ischemia injury in rats through anti-oxidative, anti-apoptotic and anti-inflammatory actions. Free Radic Biol Med. 2016 Jan;90:1–11.
18. Song K, Zhang M, Hu J, Liu Y, Liu Y, Wang Y, et al. Methane-rich saline attenuates ischemia/reperfusion injury of abdominal skin flaps in rats via regulating apoptosis level. BMC Surg. 2015 Dec;15(1).
19. Shen M, Fan D, Zang Y, Chen Y, Zhu K, Cai Z, et al. Neuroprotective effects of methane-rich saline on experimental acute carbon monoxide toxicity. J Neurol Sci. 2016 Oct;369:361–7.
20. Meng Y, Jiang Z, Li N, Zhao Z, Cheng T, Yao Y, et al. Protective Effects of Methane-Rich Saline on Renal Ischemic-Reperfusion Injury in a Mouse Model. Med Sci Monit. 2018 Oct 31;24:7794–801.
21. Li Z, Jia Y, Feng Y, Cui R, Miao R, Zhang X, et al. Methane alleviates sepsis-induced injury by inhibiting pyroptosis and apoptosis in vivo and in vitro experiments. :14.
22. Poles MZ, Bodi N, Bagyanszki M, Fekete E, Meszaros AT, Varga G, et al. Reduction of nitrosative stress by methane: Neuroprotection through xanthine oxidoreductase inhibition in a rat model of mesenteric ischemia-reperfusion. Free Radic Biol Med. 2018 May 20;120:160–9.
23. He R, Wang L, Zhu J, Fei M, Bao S, Meng Y, et al. Methane-rich saline protects against concanavalin A-induced autoimmune hepatitis in mice through anti-inflammatory and anti-oxidative pathways. Biochem Biophys Res Commun. 2016 Jan;470(1):22–8.
24. Jia Y, Li Z, Liu C, Zhang J. Methane Medicine: A Rising Star Gas with Powerful Anti-Inflammation, Antioxidant, and Antiapoptosis Properties. Oxid Med Cell Longev. 2018;2018:1–10.
25. Hauffe T, Krüger B, Bettex D et al. Shock Management For Cardio-Surgical ICU Patients — The Golden Hours. Card Fail Rev. 2015;1(2):75.
26. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol. 2000 Feb;190(3):255–66.
27. Floyd RA, Carney JM. Free radical damage to protein and DNA: Mechanisms involved and relevant observations on brain undergoing oxidative stress. Ann Neurol. 1992;32(S1):S22–7.
51
28. Bari G, Érces D, Varga G, Szűcs S, Bogáts G. A pericardialis tamponád kórélettana, klinikuma és állatkísérletes vizsgálati lehetőségei. Orv Hetil. 2018 Feb;159(5):163–7.
29. Seferović PM, Ristić AD, Imazio M, Maksimović R, Simeunović D, Trinchero R, et al. Management strategies in pericardial emergencies. Herz Kardiovaskuläre Erkrank. 2006;31(9):891–900.
30. Orbach A, Schliamser JE, Flugelman MY, Zafrir B. Contemporary evaluation of the causes of cardiac tamponade: Acute and long-term outcomes. Cardiol J. 2016 Feb 26;23(1):57–63.
31. Adler Y, Charron P, Imazio M, Badano L, Barón-Esquivias G, Bogaert J, et al. 2015 ESC Guidelines for the diagnosis and management of pericardial diseases: The Task Force for the Diagnosis and Management of Pericardial Diseases of the European Society of Cardiology (ESC)Endorsed by: The European Association for Cardio-Thoracic Surgery (EACTS). Eur Heart J. 2015 Nov 7;36(42):2921–64.
32. Kaszaki J, Nagy S, Tarnoky K, Laczi F, Vecsernyes M, Boros M. Humoral changes in shock induced by cardiac tamponade. Circ Shock. 1989 Oct;29(2):143–53.
33. Day JRS, Taylor KM. The systemic inflammatory response syndrome and cardiopulmonary bypass. Int J Surg. 2005;3(2):129–40.
34. Millar JE, Fanning JP, McDonald CI, McAuley DF, Fraser JF. The inflammatory response to extracorporeal membrane oxygenation (ECMO): a review of the pathophysiology. Crit Care. 2016 Dec;20(1).
35. Boros M, Szalay L, Kaszaki J. Endothelin-1 induces mucosal mast cell degranulation and tissue injury via ET A receptors. Clin Sci. 2002 Sep 1;103(s2002):31S-34S.
36. Szabo A, Boros M, Kaszaki J, Nagy S. The role of mast cells in mucosal permeability changes during ischemia-reperfusion injury of the small intestine. Shock Augusta Ga. 1997 Oct;8(4):284–91.
37. Boros M, Kaszaki J, Ördögh B, Nagy S. Mast cell degranulation prior to ischemia decreases ischemia-reperfusion injury in the canine small intestine. Inflamm Res. 1999 Apr;48(4):193–8.
38. Bischoff SC. Mast cells in gastrointestinal disorders. Eur J Pharmacol. 2016 May;778:139–45.
39. Krystel-Whittemore M, Dileepan KN, Wood JG. Mast Cell: A Multi-Functional Master Cell. Front Immunol. 2016 Jan 6;6.
40. González-de-Olano D, Álvarez-Twose I. Mast Cells as Key Players in Allergy and Inflammation. J Investig Allergol Clin Immunol. 2018 Dec 9;28(6):365–78.
41. Heib V, Becker M, Taube C, Stassen M. Advances in the understanding of mast cell function. Br J Haematol. 2008 Sep;142(5):683–94.
52
42. Sibilano R, Frossi B, Pucillo CE. Mast cell activation: A complex interplay of positive and negative signaling pathways. Eur J Immunol. 2014 Sep;44(9):2558–66.
43. Johnzon C-F, Rönnberg E, Pejler G. The role of mast cells in bacterial infection. Am J Pathol. 2016;186(1):4–14.
44. Rivera-Nieves J, Gorfu G, Ley K. Leukocyte adhesion molecules in animal models of inflammatory bowel disease: Inflamm Bowel Dis. 2008 Dec;14(12):1715–35.
45. Chelombitko MA, Fedorov AV, Ilyinskaya OP, Zinovkin RA, Chernyak BV. Role of reactive oxygen species in mast cell degranulation. Biochem Mosc. 2016 Dec;81(12):1564–77.
46. He Z, Ma C, Yu T, Song J, Leng J, Gu X, et al. Activation mechanisms and multifaceted effects of mast cells in ischemia reperfusion injury. Exp Cell Res. 2019 Mar;376(2):227–35.
47. Kuehn HS, Gilfillan AM. G Protein-coupled receptors and the modification of FcεRI- mediated mast cell activation. 2007;19.
48. Ito BR, Engler RL, del Balzo U. Role of cardiac mast cells in complement C5a-induced myocardial ischemia. Am J Physiol-Heart Circ Physiol. 1993 May;264(5):H1346–54.
49. Gazoni LM, Walters DM, Unger EB, Linden J, Kron IL, Laubach VE. Activation of A1, A2A, or A3 adenosine receptors attenuates lung ischemia-reperfusion injury. J Thorac Cardiovasc Surg. 2010 Aug;140(2):440–6.
50. Neurath MF. Cytokines in inflammatory bowel disease. Nat Rev Immunol. 2014 Apr 22;14(5):329–42.
51. Harrison R. Physiological Roles of Xanthine Oxidoreductase. Drug Metab Rev. 2004 Jan;36(2):363–75.
52. Xi H, Schneider BL, Reitzer L. Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage. J Bacteriol. 2000 Oct 1;182(19):5332–41.
53. Kostić DA, Dimitrijević DS, Stojanović GS, Palić IR, Đorđević AS, Ickovski JD. Xanthine Oxidase: Isolation, Assays of Activity, and Inhibition. J Chem. 2015;2015:1–8.
54. Patel NN, Toth T, Jones C, Lin H, Ray P, George SJ, et al. Prevention of post-cardiopulmonary bypass acute kidney injury by endothelin A receptor blockade*: Crit Care Med. 2011 Apr;39(4):793–802.
55. Lluch S, Moguilevsky HC, Pietra G, Shaffer AB, Hirsch LJ, Fishman AP. A reproducible model of cardiogenic shock in the dog. Circulation. 1969;39(2):205–218.
56. Starr FL 3rd, Samphilipo MAJ, White RIJ, Anderson JH. A reproducible canine model of nonocclusive mesenteric ischemia. Invest Radiol. 1982 Feb;17(1):34–6.
53
57. Jungwirth B, de Lange F. Animal Models of Cardiopulmonary Bypass: Development, Applications, and Impact. Semin Cardiothorac Vasc Anesth. 2010 Jun;14(2):136–40.
58. Fujii Y, Shirai M, Inamori S, Takewa Y, Tatsumi E. A novel small animal extracorporeal circulation model for studying pathophysiology of cardiopulmonary bypass. J Artif Organs. 2015 Mar;18(1):35–9.
59. Madrahimov N, Boyle EC, Gueler F, Goecke T, Knöfel A-K, Irkha V, et al. Novel mouse model of cardiopulmonary bypass. Eur J Cardiothorac Surg. 2018 Jan 1;53(1):186–93.
60. Tuboly E, Szabó A, Erős G, Mohácsi á, Szabó G, Tengölics R, et al. Determination of endogenous methane formation by photoacoustic spectroscopy. J Breath Res. 2013 Nov 1;7(4):046004.
61. Kuebler WM, Abels C, Schuerer L. Measurement of neutrophil content in brain and lung tissue by a modified myeloperoxidase assay. Nt J Microcirc Clin Exp. 1996;(16):89–97.
62. Beckman JS, Parks DA, Pearson JD, Marshall PA, Freeman BA. A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues. Free Radic Biol Med. 1989 Jan;6(6):607–15.
63. Zimmermann T, Schuster R, Lauschke G, Trausch M, Albrecht S, Kopprasch S, et al. Chemiluminescence response of whole blood and separated blood cells in cases of experimentally induced pancreatitis and MDTQ-DA—Trasylol—ascorbic acid therapy. Anal Chim Acta. 1991 Dec;255(2):373–81.
64. Kovács T, Varga G, Érces D, Tőkés T, Tiszlavicz L, Ghyczy M, et al. Dietary Phosphatidylcholine Supplementation Attenuates Inflammatory Mucosal Damage in a Rat Model of Experimental Colitis: Shock. 2012 Aug;38(2):177–85.
65. Érces D, Nógrády M, Nagy E, Varga G, Vass A, Süveges G, et al. Complement C5A Antagonist Treatment Improves the Acute Circulatory and Inflammatory Consequences of Experimental Cardiac Tamponade: Crit Care Med. 2013 Nov;41(11):e344–51.
66. Vass A, Süveges G, Érces D, Nógrády M, Varga G, Földesi I, et al. Inflammatory Activation after Experimental Cardiac Tamponade. Eur Surg Res. 2013;51(1–2):1–13.
67. Iselin CE, Ny L, Mastrangelo D, Felley-Bosco E, Larsson B, Alm P, et al. The nitric oxide pathway in pig isolated calyceal smooth muscle. Neurourol Urodyn. 1999;18(6):673–85.
68. Bari G, Szűcs S, Érces D et al. Experimental pericardial tamponade–translation of a clinical problem to its large animal model. Turk J Surg. 2018 Sep 28;34(3):205–11.
69. Bari G, Szűcs S, Érces D, Ugocsai M, Bozsó N, Balog D, et al. A cardiogen sokk modellezése pericardialis tamponáddal. Magy Seb. 2017 Dec;70(4):297–302.
54
70. Wang L, Yao Y, He R, Meng Y, Li N, Zhang D, et al. Methane ameliorates spinal cord ischemia-reperfusion injury in rats: Antioxidant, anti-inflammatory and anti-apoptotic activity mediated by Nrf2 activation. Free Radic Biol Med. 2017 Feb;103:69–86.
71. Strifler G, Tuboly E, Szel E, Kaszonyi E, Cao C, Kaszaki J, et al. Inhaled Methane Limits the Mitochondrial Electron Transport Chain Dysfunction during Experimental Liver Ischemia-Reperfusion Injury. PloS One. 2016;11(1):e0146363.
72. Nguyen BAV, Fiorentino F, Reeves BC, Baig K, Athanasiou T, Anderson JR, et al. Mini Bypass and Proinflammatory Leukocyte Activation: A Randomized Controlled Trial. Ann Thorac Surg. 2016 Apr;101(4):1454–63.
73. McDonald CI, Fraser JF, Coombes JS, Fung YL. Oxidative stress during extracorporeal circulation. Eur J Cardiothorac Surg. 2014 Dec 1;46(6):937–43.
74. Zakkar M, Guida G, Suleiman M-S, Angelini GD. Cardiopulmonary Bypass and Oxidative Stress. Oxid Med Cell Longev. 2015;2015:1–8.
75. Whitlock RP, Devereaux PJ, Teoh KH, Lamy A, Vincent J, Pogue J, et al. Methylprednisolone in patients undergoing cardiopulmonary bypass (SIRS): a randomised, double-blind, placebo-controlled trial. The Lancet. 2015 Sep;386(10000):1243–53.
76. Bernardi MH, Rinoesl H, Dragosits K, Ristl R, Hoffelner F, Opfermann P, et al. Effect of hemoadsorption during cardiopulmonary bypass surgery – a blinded, randomized, controlled pilot study using a novel adsorbent. Crit Care. 2016 Dec;20(1).
77. Vogel PA, Yang X, Moss NG, Arendshorst WJ. Superoxide Enhances Ca 2+ Entry Through L-Type Channels in the Renal Afferent Arteriole. Hypertension. 2015 Aug;66(2):374–81.
78. Pacher P. Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol. Pharmacol Rev. 2006 Mar 1;58(1):87–114.
79. Murphy GJ, Lin H, Coward RJ, Toth T, Holmes R, Hall D, et al. An initial evaluation of post-cardiopulmonary bypass acute kidney injury in swine☆. Eur J Cardiothorac Surg. 2009 Nov;36(5):849–55.
80. Bellomo R, Giantomasso DD. Noradrenaline and the kidney: friends or foes? Crit Care Med.2001;5(6):5.
81. Schaer GL, Fink MP, Parrillo JE. Norepinephrine alone versus norepinephrine plus low-dose dopamine: enhanced renal blood flow with combination pressor therapy. Crit Care Med. 1985 Jun;13(6):492–6.
82. Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V. The pig: a model for human infectious diseases. Trends Microbiol. 2012 Jan;20(1):50–7.
55
83. Janssen M, van der Meer P, de Jong JW. Antioxidant defences in rat, pig, guinea pig, and human hearts: comparison with xanthine oxidoreductase activity. Cardiovasc Res. 1993 Nov;27(11):2052–7.
56
8. ACKNOWLEDGEMENTS
I would like to express my gratitude to Professor Mihály Boros, Head of the
Institute of Surgical Research, for granting me the opportunity to work in his
department and for his scientific guidance.
I am also grateful to Gábor Bogáts, the Head of the Department of Cardiac
Surgery, for his support in my scientific work.
I am especially grateful to my two supervisors, Gabriella Varga and Dániel
Érces, for their continuous support and for introducing me to the world of scientific
problems. Without their continuous work and never-ending encouragement, this thesis
would never have been written.
I wish to express my thanks for the excellent technical assistance at the Institute
of Surgical Research and for assistance I received from Ágnes Fekete, Csilla Mester,
Nikolett Beretka, Éva Nagyiván, Andrea Bús, Ágnes Lilla Kovács, Péter Szabó, Károly
Tóth and Péter Sárkány.
I would like to thank my family and my parents for their love, patience and trust.
The PhD thesis was funded by Hungarian National Research, Development and
Innovation Office NKFIH-K120232 and NKFIH-K116861, IKOM GINOP-2.3.2-15-
2016-00015 and UNKP 20391-3/ 2018 /FEKUSTRAT grants.
57
9. ANNEX